Our long-term goal is to clarify the link between inflammation and cancer in general, and to prevent colitis associated cancer (CAC) in particular. As CAC pathogenesis is unclear, our immediate objective is to determine mechanisms. Therefore, for CAC we will determine the roles of: (1) gene defects, (2) cancer stem cells (CSCs), especially their clonality and recapitulation of human colorectal cancer, and (3) stromal fibroblasts, especially in the generation of colon cancer initiating cells (CCICs). For interrogating these relationships, CCICs and the colitic stroma are powerful tools while spheroid cultures (spheres) and murine xenografts are powerful cancer models. Our central hypothesis is that the above three factors are key to CAC development. The proposed research is relevant to the NIH's mission to develop fundamental knowledge that will potentially help reduce the burdens of human disability and disease. Our preliminary data demonstrate our ability to (1) enrich for precursor CCICs (pCCICs) in a fraction of non-dysplastic colon from human ulcerative colitis (UC); (2) perpetuate pCCIC-derived tumors in mice; (3) maintain pCCICs as spheres. We found that colitic stromal fibroblasts possess a cytokine and signaling profile similar to those fibroblasts associated with CRC. We will test our central hypothesis using three aims. Aim 1) To determine if gene defects acquired by the xenografts and spheres from UC and from CRC follow those in the classical adenoma-to-carcinoma pathway. We will measure the expanded distribution of a putative colon stem cell marker, ALDH, and use this marker to enrich for epithelium undergoing self-renewal. We will use the mutational profile to predict the success of UC-derived epithelium in xenograft and sphere assays. Aim 2) To determine if spheres from colon cancer initiating cells are true CSC which mimic the clonal and metastatic behavior of human CRC. Aim 2 focuses on the spheres themselves. We will examine the pCCICs for self-renewal via clonogenic, differentiation assays. Aim 3) To show the key contribution of IL8 / UC stroma to epithelial proliferation. Fibroblast isolates from normal, colitic, and cancer tissues will be placed in co-culture with pCCICs to determine their effects on both proliferation and tumorigenicity. Innovation: Our novel cellular tools including pCCICs, inflamed stroma, ALDH, a specific SC/CSC marker, and cancer models - spheres & xenografts - derived from human tissues (patients with normal, colitic, and cancerous colon). Significance: Our studies will (i) define the tumor initiating colonic epithelial cell in a preneoplastic, inflammatory state, (ii) reveal epithelial/stromal relationships, (iii) clarify the link between inflammation and cancer, (iv) facilitate the development of novel strategies to prevent CAC, and (v) substantially improve our ability to predict those patients who will progress to malignancy.